Purpose:
Inflammasomes have remerged as key players in the innate immune system. The NLRC4/NAIP5 inflammasome complex acts as an intracellular sensor that detects flagellin and mounts an inflammatory response that includes pyroptosis, activation of caspase 1, cleavage of IL-1 β and IL-18. We recently discovered a novel early and critical effector function of inflammasome activation, namely the rapid formation of an eicosanoid storm by resident macrophages. In the eye, inflammasomes have been implicated in the progression of age-related macular degeneration; however its role in the ocular surface still remains to be explored. Hence, we investigated the effects of NLRC4/NAIP5 activation in the ocular surface during an acute inflammatory/reparative response.

Methods:
MyD88/Trif KO, NLRC4/NAIP5 KO and C57BJ/6 wild type mice were used to directly assess the role the inflammasome. A fusion protein (FlaTox) consisting of flagellin attached to the pore-forming, amino-terminal domain of the B. anthracis lethal factor was topically applied to the eye following full corneal epithelial abrasion. The control group received an inactive mutant construct (AAA). Wound healing was measured with ImagePro Express and inflammation assessed by leukocyte infiltration by enzyme assays, FACS and immunohistochemistry. Eicosanoid formation was quantified using LC/MS/MS-based lipidomics and gene expression by QPCR.

Results:
NLRC4 and NAIP5 are expressed and functional in the ocular surface. 4 days after injury, MyD88/Trif KO corneas that received FlaTox exhibited markedly decreased wound healing (1.8-fold greater epithelial defect) when directly compared to the control AAA group, consistent with pronounced corneal opacity, inflammatory neovascularization, and greater neutrophil infiltration. Targeted inflammasome activation triggered an early eicosanoid storm in the conjunctiva, which was abrogated in NLRC4/NAIP5 KO mice.

Conclusions:
These findings provide the first evidence for a role of the NLRC4/NAIP5 inflammasome in ocular surface innate immune responses. NLRC4/NAIP5 activation lead to the rapid formation of an eicosanoid storm and triggered a far reaching and perilous immune response, which can have severe ramifications to ocular health. The molecular mechanisms and regulation of inflammasomes are of great interest to the health of the visual axis and remain to be elucidated.